CRISPR technology has transformed biology. Genome-wide loss-of-function (LoF) phenotypic screens using CRISPR-based genomic libraries provide extremely powerful ways to identify novel mediators of biological pathways by systematically knocking out individual genes in cells.
The CTS Drug and Target Discovery High Throughput Screening Facility (DTD-HTSF) now offers screens of arrayed sgRNA CRISPR genomic libraries that allow for the identification of genes and pathways that contribute to a phenotype. sgRNAs that target a single gene (3 sgRNAs/gene) are arrayed in individual wells of microplates. The sgRNAs can be delivered to cells using three mechanisms: (1) transfected as sgRNA/Cas9 RNPs, (2) transfected with RNA-Cas9 or (3) transfected into Cas9 expressing cells. Phenotypic outcomes are measured to determine involvement of each gene in a phenotype of interest. Practically any phenotype can be measured using detection tools available at the DTD-HTSF facility.
CTS currently has available for investigators the following arrayed sgRNA CRISPR libraries from Synthego:
- Human arrayed whole genome sgRNA CRISPR library: 19753 genes
- Mouse arrayed whole genome sgRNA CRISPR library: 20914 genes
- Human autophagy/mTOR arrayed sgRNA CRISPR library: 491 genes
- The following gene subsets can be generated for screening: kinases, GPCRs, epigenetic modifiers, transcription factors, nuclear hormone receptors, tumor suppressors, cell cycle, druggable targets, ubiquitin ligases (E1, E2, E3), deubiquitinating enzymes
- Custom gene subsets can be easily generated.
CTS will provide the following services for the completing of each screen: (1) optimization of transfection arrayed sgRNAs using either cells expressing spCas9, formation of ribonucleotide particles with sgRNAs and recombinant spCas9 or cotransfection of sgRNAs with mRNA-Cas9; (2) optimization of screening formats that can include cell viability or high content imaging; performing the screens and (4) data analysis and hit identification.
Dr. Matthew Hart, Director of CTS and of the DTD-HTSF will provide consultation and guidance in performing CRISPR-based genomic screens. Interested investigators should contact Dr. Hart (matthew-j-hart@ouhsc.edu) to discuss project feasibility and development.